二甲双胍通过CXCR4/Akt信号通路增强结肠癌细胞对5-FU敏感性的实验研究  

Experimental study on metformin enhancing the sensitivity of colon cancer cells to 5-FU through CXCR4/Akt signaling pathway

作　　者：严虹霞[1] 何林秀 蔡新凤 张毅勋[2] Yan Hongxia;He Linxiu;Cai Xinfeng;Zhang Yixun(Department of Pharmacy,Shanxi Province Cancer Hospital,Shanxi Hospital Affiliated to Cancer Hospital,Chinese Academy of Medical Sciences,Cancer Hospital Affiliated to Shanxi Medical University,Taiyuan 030013,China;Department of Colorectal Surgery,Shanxi Province Cancer Hospital,Shanxi Hospital Affiliated to Cancer Hospital,Chinese Academy of Medical Sciences,Cancer Hospital Affiliated to Shanxi Medical University,Taiyuan 030013,China)

机构地区：[1]山西省肿瘤医院、中国医学科学院肿瘤医院、山西医院山西医科大学附属肿瘤医院药学部,太原030013 [2]山西省肿瘤医院、中国医学科学院肿瘤医院山西医院、山西医科大学附属肿瘤医院结直肠外科,太原030013

出　　处：《肿瘤研究与临床》2024年第12期919-927,共9页Cancer Research and Clinic

基　　金：山西省科技厅自然科学研究面上项目(202203021221280);山西省卫生健康委员会一般项目(2020063)。

摘　　要：目的探讨二甲双胍对结肠癌细胞5-氟尿嘧啶(5-FU)化疗敏感性的影响及其可能的作用机制。方法体外培养结肠癌细胞株HCT-116和HT-29,采用CCK-8法测定不同浓度(1.25、2.5、5、10、20、40、80 mmol/L)盐酸二甲双胍(MET-HCl)或不同浓度(1.25、2.5、5、10、20、40μmol/L)5-FU干预后细胞活力,计算细胞存活率和48 h两药半数抑制浓度(IC_(50))。通过Chou-Talalay模型计算MET-HCl与5-FU配伍的联合指数(CI),以抑制率(Fa)=0.5时MET-HCl、5-FU浓度确定后续实验中两药的浓度。采用CCK-8法和Annexin V-FITC/PI流式细胞术分别检测实验浓度下两药单独和联合作用的HCT-116细胞或HT-29细胞的存活率和凋亡率,以加入不含药物培养液的细胞为对照组。HCT-116细胞、HT-29细胞中转染CXCR4特异性小干扰RNA(siRNA),通过蛋白质印迹法检测CXCR4蛋白表达水平下降(P<0.05),提示CXCR4敲低成功。采用CCK-8法检测两药单独及联合作用的敲低CXCR4表达的细胞存活率,以加入不含药物培养液的未经转染的细胞为si-对照组。蛋白质印迹法检测两药单独及联合作用的HCT-116细胞或HT-29细胞中CXCR4/Akt信号通路相关蛋白的表达情况。结果CCK-8法检测显示,MET-HCl和5-FU分别以浓度-时间依赖的方式减弱HCT-116和HT-29细胞活力,作用48 h时HCT-116细胞MET-HCl和5-FU的IC_(50)分别为(13.0±5.8)mmol/L、(16.9±7.2)μmol/L,HT-29细胞分别为(8.6±2.8)mmol/L、(9.7±3.1)μmol/L。CCK-8法测定HCT-116细胞或HT-29细胞用药48 h后细胞抑制率为50%、75%及90%时,HCT-116细胞对应的CI值分别为0.48、0.37、0.25,HT-29细胞分别为0.57、0.51、0.49,提示两药联用具有协同效应。按Fa=0.5,确定MET-HCl、5-FU实验浓度分别为10 mmol/L、5μmol/L。CCK-8法检测显示,10 mmol/L MET-HCl、5μmol/L 5-FU单独或二者联合作用HCT-116细胞或HT-29细胞48 h后,各用药组细胞存活率均低于对照组(均P<0.05),单独应用MET-HCl与单独应用5-FU的细胞间存活率差异均无统Objective To investigate the effect of metformin on the chemosensitivity of colon cancer cells to 5-fluorouracil(5-FU)and its possible mechanism.Methods Colon cancer cell lines HCT-116 and HT-29 were cultured in vitro,and the CCK-8 method was used to determine cell viability after intervention with different concentrations(1.25,2.5,5,10,20,40,80 mmol/L)of metformin hydrochloride(MET-HCl)or different concentrations(1.25,2.5,5,10,20,40μmol/L)of 5-FU.The survival rate and 48-hour half maximal inhibitory concentration(IC_(50))of the two drugs were calculated.The combined index(CI)of MET-HCl and 5-FU was calculated using the Chou-Talalay model,and the concentrations of MET-HCl and 5-FU in subsequent experiments were determined based on the concentration of the two drugs when the inhibition rate(Fa)was 0.5.The CCK-8 method and Annexin V-FITC/PI flow cytometry were used to detect the survival rate and apoptosis rate of HCT-116 cells or HT-29 cells treated with the two drugs alone and in combination at experimental concentrations,respectively.Cells treated with drug free culture flnid were used as the control group.CXCR4 specific small interfering RNA(siRNA)was transfected into HCT-116 cells and HT-29 cells,and the decreased(P<0.05)expression of CXCR4 protein detected by Western blotting indicated successful knockdown of CXCR4.CCK-8 method was used to detect the survival rate of cells with CXCR4 knockdown by two drugs alone and in combination,and the untransfected cells added drug free culture fluid were served as the si-control group.Western blotting was used to detect the expression of proteins related to the CXCR4/Akt signaling pathway in HCT-116 cells or HT-29 cells treated with two drugs alone or in combination.Results CCK-8 assay showed that MET-HCl and 5-FU decreased the viability of HCT-116 and HT-29 cells in a concentration-time-dependent manner;at 48 h,IC_(50) of MET-HCl and 5-FU in HCT-116 cells were(13.0±5.8)mmol/L and(16.9±7.2)μmol/L,respectively,and IC_(50) in HT-29 cells were(8.6±2.8)mmol/L and(9.7±3

关 键 词：结直肠肿瘤 二甲双胍 氟尿嘧啶 受体 CXCR4 X-射线修复交叉互补蛋白1 信号传导 

分 类 号：R73[医药卫生—肿瘤]